Identification of receptor genes in renal cell carcinoma associated with angiogenesis by differential hybridization technique

To screen the receptor genes in renal cell carcinoma (RCC) associated with angiogenesis, we performed differential hybridization of the cDNA library of membrane-type protein tyrosine kinases (mPTKs). Three thousand plaques of a mPTKs-enriched cDNA library were screened with mPTKs mixture probes produced from hypervascular RCC tissues and RCC cell lines. Six different cDNA fragments of the PTK genes were isolated, and the sequence analysis showed that these represented cDNAs for TIE1, KDR, FMS, FGFR-4, JAK1 and HCK. Of these genes, the expression of TIE1, KDR, and FGFR-4 was studied in RCC tissue and cell lines by Northern blot analysis. We also investigated the expression of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptor FLT-1. In all the hypervascular RCC tissues, the amounts of mRNAs for KDR and FLT-1 were increased compared to adjacent normal tissues. The TIE1 and FGFR-4 genes were also overexpressed in most of the hypervascular RCC tissues, while no mRNA of KDR, FLT-1, or TIE1 could be detected in any of the four human RCC cell lines. The amounts of the VEGF and PlGF mRNAs were increased in hypervascular RCC tissues, while VEGF mRNA was detected in the four cell lines but PlGF mRNA was not. FGFR-4 mRNA was expressed in three of the four cell lines. These results suggest that KDR, FLT-1, PlGF and TIE1 mRNAs are present in the mesenchymal cells of RCC, while VEGF and FGFR-4 genes are expressed in RCC cells themselves in vivo.     

Expression of immunological markers on leukemic cells before and after cryopreservation and thawing

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.     

FLT-3: a new focus in the understanding of acute leukemia

The FMS-like tyrosine kinase-3 (FLT-3), which belongs to the class III receptor tyrosine kinase family, is primarily expressed by hematopoietic cells and plays an important role in hematopoiesis. FLT-3 is also expressed in the majority of acute leukemias, in which the presence of FLT-3 activating mutations is associated with poor prognosis. Consequently, there has been a recent surge in the development of FLT-3 inhibitors for the molecular targeting of leukemia, and many of these are now in clinical trials. An improved understanding of how FLT-3 interacts with its ligand, as well as how FLT-3 activating mutations are able to trigger downstream intracellular signaling pathways, will provide greater insight to how small molecule inhibitors may best be utilized and combined with established chemotherapeutic drugs for the management of patients with high-risk acute leukemia.     

Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.     

Placenta growth factor-1 exerts time-dependent stabilization of adherens junctions following VEGF-induced vascular permeability

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.     

Modification of a promiscuous inhibitor shifts the inhibition from γ-secretase to FLT-3

The inhibition of FLT-3 activity is an interesting target for the treatment of acute myeloid leukemia (AML). The serendipitous identification of FLT-3 inhibitors from a CK1/γ-secretase programme provided compounds with dual inhibitory activity. We analyzed the structure–activity relationship of these inhibitors and derivatized them to arrive at compounds with reduced impact on γ-secretase activity and enhanced FLT-3 inhibition.     

Biotinylated theta-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.     

AML1-ETO interacts with Sp1 and antagonizes Sp1 transactivity through RUNT domain

AML1-ETO fusion protein is observed in approximately 12% of acute myeloid leukemia. In the present research, we found that AML1-ETO is able to inhibit Sp1 transactivity. We also found that this inhibition of Sp1 transactivity by AML1-ETO is achieved by interaction between Sp1 and RUNT domain of AML1. AML1b is able to abrogate the inhibition of AML1-ETO. Since Sp1 is involved in hematopoietic cell differentiation, we proposed that AML1-ETO promotes leukemogenesis by blocking cell differentiation through inhibition of Sp1 transactivity.     

The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.     

Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1.In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells.     

The crystal structure of placental growth factor in complex with domain 2 of vascular endothelial growth factor receptor-1

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and plays an important role in pathological angiogenic events. PlGF exerts its biological activities through binding to VEGFR1, a receptor tyrosine kinase that consists of seven immunoglobulin-like domains in its extracellular portion. Here we report the crystal structure of PlGF bound to the second immunoglobulin-like domain of VEGFR1 at 2.5 A resolution and compare the complex to the closely related structure of VEGF bound to the same receptor domain. The two growth factors, PlGF and VEGF, share a sequence identity of approximately 50%. Despite this moderate sequence conservation, they bind to the same binding interface of VEGFR1 in a very similar fashion, suggesting that both growth factors could induce very similar if not identical signaling events.     

High serum levels of soluble interleukin-2 receptor in acute myeloid leukemia: Correlation with poor prognosis and CD4 expression on blast cells

Backgorund: Although increased serum levels of soluble interleukin-2 receptor (sIL-2R) and their clinical importance are well known in mature type lymphoproliferative disorders (LD), little data is available about such information in acute type hematological malignancies. Methods: We examined the serum levels of sIL-2R in 57 adult patients with acute type leukemias: 32 with acute myeloid leukemia (AML), 14 acute lymphoblastic leukemia (ALL) and 11 chronic myelocytic leukemia in blast crisis (CMLBC), and in 29 adult patients with mature type LD, and assessed their cellular and clinical relevance in acute type leukemias. Results: No significant differences were seen in the sIL-2R levels between acute type leukemias and mature type LD. In AML, serum sIL-2R levels were related to the cell surface CD4 expression on blast cells, and patients with higher levels ≧2000U/ml had a poorer prognosis (lower response to chemotherapy and shorter overall survival). Conclusions: These results suggest that serum sIL-2R level elevates in acute type leukemias like mature type LD, and increased sIL-2R levels in adult AML are correlated with certain biological and clinical characteristics.     

Angiotensin II upregulates the expression of placental growth factor in human vascular endothelial cells and smooth muscle cells

Background  

Atherosclerosis is now recognized as a chronic inflammatory disease. Angiotensin II (Ang II) is a critical factor in inflammatory responses, which promotes the pathogenesis of atherosclerosis. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family cytokines and is associated with inflammatory progress of atherosclerosis. However, the potential link between PlGF and Ang II has not been investigated. In the current study, whether Ang II could regulate PlGF expression, and the effect of PlGF on cell proliferation, was investigated in human vascular endothelial cells (VECs) and smooth muscle cells (VSMCs).     

Formation of amyloids by Abeta-(1-42) on NGF-differentiated PC12 cells: roles of gangliosides and cholesterol

The conversion of soluble, non-toxic amyloid beta-protein (Abeta) to aggregated, toxic Abeta could be the key step in the development of Alzheimer's disease. Liposomal studies have proposed that Abeta-(1-40) preferentially recognizes a cholesterol-dependent cluster of gangliosides and a conformationally altered form of Abeta promotes the aggregation of the protein. Cell experiments using fluorescein-labeled Abeta-(1-40) supported this model. Here, the interaction of native Abeta-(1-42) with unfixed rat pheochromocytoma PC12 cells was visualized using the amyloid-specific dye Congo red. Abeta-(1-42) preferentially bound to ganglioside and cholesterol-rich domains of cell membranes and formed amyloids in a time-dependent manner. These observations corroborate the model involving ganglioside-mediated accumulation of Abeta. The NGF-induced differentiation of PC12 cells into neuron-like cells caused a marked increase in both gangliosides and cholesterol, and thereby greatly potentiated the accumulation and cytotoxicity of Abeta-(1-42). NGF-differentiated cells exposed to Abeta-(1-42) had degenerated neurites, in which ganglioside and cholesterol-rich domains were localized, preceding cell death. A reduction in the amount of cholesterol by the cholesterol synthesis inhibitor compactin almost nullified the formation of amyloids by Abeta-(1-42). Our system using NGF-differentiated PC12 cells and Congo red is useful for screening inhibitors of the formation of amyloids by and cytotoxicity of Abeta.